RESUMEN
Toll-like receptor (TLR) agonists can act as immune stimulants alone or as part of alum or oil formulations. Humoral and cellular immune responses were utilized to assess quantitative and qualitative immune response enhancement by TLR agonists using recombinant protective antigen (rPA) of B. anthracis as a model antigen. To rPA, combined with aluminum hydroxide (Alhydrogel; Al(OH)3) or squalene (AddaVax™), was added one of 7 TLR agonists: TLR2 agonist Pam3CysSK4 (PamS), TLR3 agonist double stranded polyinosinic:polycytidylic acid (PolyIC), TLR4 agonists Monophosphoryl lipid A (MPLA) or glucopyranosyl lipid A (GLA), TLR7-8 agonists 3M-052 or Resiquimod (Resiq), or TLR9 agonist CPG 7909 (CPG). CD-1 or BALB/c mice received two intraperitoneal or intramuscular immunizations 14 days apart, followed by serum or spleen sampling 14 days later. All TLR agonists except PamS induced high levels of B. anthracis lethal toxin-neutralizing antibodies and immunoglobulin G (IgG) anti-PA. Some responses were >100-fold higher than those without a TLR agonist, and IP delivery (0.5 mL) induced higher TLR-mediated antibody response increases compared to IM delivery (0.05 mL). TLR7-8 and TLR9 agonists induced profound shifts of IgG anti-PA response to IgG2a or IgG2b. Compared to the 14-day immunization schedule, use of a shortened immunization schedule of only 7 days between prime and boost found that TLR9 agonist CPG in a squalene formulation maintained higher interferon-γ-positive cells than TLR4 agonist GLA. Variability in antibody responses was lower in BALB/c mice than CD-1 mice but antibody responses were higher in CD-1 mice. Lower serum 50% effective concentration (EC50) values were found for rPA-agonist formulations and squalene formulations compared to Al(OH)3 formulations. Lower EC50 values also were associated with low frequency detection of linear peptide epitopes. In summary, TLR agonists elicited cellular immune responses and markedly boosted humoral responses.
Asunto(s)
Bacillus anthracis , Adyuvantes Inmunológicos , Hidróxido de Aluminio , Animales , Antígenos , Inmunoglobulina G , Ratones , Ratones Endogámicos BALB C , Escualeno , Receptor Toll-Like 2 , Receptor Toll-Like 4/agonistas , Receptor Toll-Like 7/agonistas , Receptor Toll-Like 9/agonistasRESUMEN
AV7909 is a next-generation anthrax vaccine under development for post-exposure prophylaxis following suspected or confirmed Bacillus anthracis exposure, when administered in conjunction with the recommended antibacterial regimen. AV7909 consists of the FDA-approved BioThrax® vaccine (anthrax vaccine adsorbed) and an immunostimulatory Toll-like receptor 9 agonist oligodeoxynucleotide adjuvant, CPG 7909. The purpose of this study was to evaluate the potential systemic and local toxicity of AV7909 when administered via repeat intramuscular injection to the right thigh muscle (biceps femoris) to male and female Sprague Dawley rats. The vaccine was administered on Days 1, 15, and 29 and the animals were assessed for treatment-related effects followed by a 2-week recovery period to evaluate the persistence or reversibility of any toxic effects. The AV7909 vaccine produced no apparent systemic toxicity based on evaluation of clinical observations, body weights, body temperature, clinical pathology, and anatomic pathology. Necrosis and inflammation were observed at the injection sites as well as in regional lymph nodes and adjacent tissues and were consistent with immune stimulation. Antibodies against B. anthracis protective antigen (PA) were detected in rats treated with the AV7909 vaccine, confirming relevance of this animal model for the assessment of systemic toxicity of AV7909. In contrast, sera of rats that received saline or soluble CPG 7909 alone were negative for anti-PA antibodies. Overall, 3 intramuscular immunizations of Sprague Dawley rats with AV7909 were well tolerated, did not induce mortality or any systemic adverse effects, and did not result in any delayed toxicity.
Asunto(s)
Adyuvantes Inmunológicos/administración & dosificación , Vacunas contra el Carbunco/administración & dosificación , Oligodesoxirribonucleótidos/administración & dosificación , Adyuvantes Inmunológicos/toxicidad , Animales , Vacunas contra el Carbunco/toxicidad , Anticuerpos Antibacterianos/sangre , Anticuerpos Neutralizantes/sangre , Antígenos Bacterianos/inmunología , Toxinas Bacterianas/inmunología , Femenino , Reacción en el Punto de Inyección/sangre , Reacción en el Punto de Inyección/etiología , Reacción en el Punto de Inyección/inmunología , Reacción en el Punto de Inyección/patología , Inyecciones Intramusculares , Masculino , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/patología , Oligodesoxirribonucleótidos/toxicidad , Profilaxis Posexposición , Ratas Sprague-DawleyRESUMEN
The anthrax vaccine candidate AV7909 is being developed as a next-generation vaccine for post-exposure prophylaxis (PEP) against inhalational anthrax. In clinical studies, two vaccinations with AV7909 administered either two or four weeks apart induced an enhanced immune response compared to BioThrax® (Anthrax Vaccine Adsorbed) (AVA). Anthrax toxin-neutralizing antibody (TNA) levels on Day 70 following initial vaccination that were associated with protection of animals exposed to inhalational anthrax were previously reported for the 0, 4-week AV7909 vaccination regimen. The current study shows that a 0, 2-week AV7909 vaccination regimen protected guinea pigs (GPs) and nonhuman primates (NHPs) against a lethal inhalational anthrax challenge on Days 28 and 70 after the first immunization. An earlier induction of protective TNA levels using a 0, 2-week AV7909 vaccination regimen may provide benefit over the currently approved AVA PEP 0, 2, and 4-week vaccination regimen.
Asunto(s)
Vacunas contra el Carbunco , Carbunco , Bacillus anthracis , Animales , Carbunco/prevención & control , Anticuerpos Antibacterianos , Anticuerpos Neutralizantes , Antígenos Bacterianos , Cobayas , Profilaxis Posexposición , PrimatesRESUMEN
A next-generation anthrax vaccine candidate, AV7909, is being developed for post-exposure prophylaxis (PEP) of inhalational anthrax in combination with the recommended course of antimicrobial therapy. Clinical efficacy studies of anthrax countermeasures in humans are not ethical or feasible, therefore, licensure of AV7909 for PEP is being pursued under the US Food and Drug Administration (FDA) Animal Rule, which requires that evidence of effectiveness be demonstrated in an animal model of anthrax, where results of studies in such a model can establish reasonable likelihood of AV7909 to produce clinical benefit in humans. Initial development of a PEP model for inhalational anthrax included evaluation of post-exposure ciprofloxacin pharmacokinetics (PK), tolerability and survival in guinea pigs treated with various ciprofloxacin dosing regimens. Three times per day (TID) intraperitoneal (IP) dosing with 7.5 mg/kg of ciprofloxacin initiated 1 day following inhalational anthrax challenge and continued for 14 days was identified as a well tolerated partially curative ciprofloxacin treatment regimen. The added benefit of AV7909 vaccination was evaluated in guinea pigs given the partially curative ciprofloxacin treatment regimen. Groups of ciprofloxacin-treated guinea pigs were vaccinated. 1 and 8 days post-challenge with serial dilutions of AV7909, a 1:16 dilution of AVA, or normal saline. A group of untreated guinea pigs was included as a positive control to confirm lethal B. anthracis exposure. Post-exposure vaccination with the AV7909 anthrax vaccine candidate administered in combination with the partially curative ciprofloxacin treatment significantly increased survival of guinea pigs compared to ciprofloxacin treatment alone. These results suggest that the developed model can be useful in demonstrating added value of the vaccine for PEP.
Asunto(s)
Vacunas contra el Carbunco/administración & dosificación , Carbunco , Modelos Animales de Enfermedad , Profilaxis Posexposición , Infecciones del Sistema Respiratorio , Animales , Carbunco/prevención & control , Antibacterianos/farmacocinética , Ciprofloxacina/farmacocinética , Cobayas , Infecciones del Sistema Respiratorio/prevención & controlRESUMEN
The anthrax vaccine candidate AV7909 is being developed as a next-generation vaccine for a post-exposure prophylaxis (PEP) indication against anthrax. AV7909 consists of the anthrax vaccine adsorbed (AVA) (Emergent BioSolutions Inc., Lansing, MI) bulk drug substance adjuvanted with the immunostimulatory oligodeoxynucleotide (ODN) compound, CPG 7909. The addition of CPG 7909 to AVA enhances both the magnitude and the kinetics of antibody responses in animals and human subjects, making AV7909 a suitable next-generation vaccine for use in a PEP setting. Emergent has produced a thermostable (lyophilized) formulation of AV7909 vaccine utilizing drying technology. The purpose of the study described here was to assess the immunogenicity and efficacy of the lyophilized formulation of the AV7909 vaccine candidate as compared with the liquid formulation in the guinea pig general-use prophylaxis (GUP) model. The study also provides initial information on the relationship between the immune response induced by the thermostable formulation of the vaccine, as measured by the toxin neutralization assay (TNA), and animal survival following lethal anthrax aerosol challenge. Results demonstrated that there were no significant differences in the immunogenicity or efficacy of lyophilized AV7909 against lethal anthrax spore aerosol challenge in the guinea pig model as compared to liquid AV7909. For both vaccine formulations, logistic regression modeling showed that the probability of survival increased as the pre-challenge antibody levels increased.
Asunto(s)
Vacunas contra el Carbunco/química , Vacunas contra el Carbunco/inmunología , Anticuerpos Antibacterianos/sangre , Inmunogenicidad Vacunal , Temperatura , Adyuvantes Inmunológicos , Animales , Carbunco/prevención & control , Anticuerpos Neutralizantes/sangre , Antígenos Bacterianos/inmunología , Femenino , Liofilización , Cobayas , Masculino , Oligodesoxirribonucleótidos/inmunología , Profilaxis Posexposición , Vacunación , Potencia de la VacunaRESUMEN
Most of our understanding of the development and phenotype of alternatively activated macrophages (AAMs) has been obtained from studies investigating the response of bone marrow- and peritoneal-derived cells to IL-4 or IL-13 stimulation. Comparatively little is known about the development of AAMs in the lungs, and how the complex signals associated with pulmonary inflammation influence the AAM phenotype. Here, we use Nippostrongylus brasiliensis to initiate AAM development and define the dynamics of surface molecules, gene expression, and cell function of macrophages isolated from lung tissue at different times postinfection (PI). Initially, lung macrophages take on a foamy phenotype, up-regulate MHC and costimulatory molecules, express reduced levels of TNF and IL-12, and undergo proliferation. Cells isolated between days 8 and 15 PI adopt a dense, granular phenotype and exhibit reduced levels of costimulatory molecules and elevated levels of programmed death ligand-1 (PDL-1) and PDL-2 and an increase in IL-10 expression. Functionally, AAMs isolated on days 13-15 PI demonstrate an enhanced capacity to take up and sequester antigen. However, these same cells did not mediate antigen-specific T cell proliferation and dampened the proliferation of CD3/CD28-activated CD4+ T cells. These data indicate that the alternative activation of macrophages in the lungs, although initiated by IL-4/IL-13, is a dynamic process that is likely to be influenced by other immune and nonimmune factors in the pulmonary environment.
Asunto(s)
Activación de Macrófagos/inmunología , Macrófagos Alveolares/parasitología , Nippostrongylus/patogenicidad , Infecciones por Strongylida/parasitología , Animales , Líquido del Lavado Bronquioalveolar/química , Antígenos CD28/genética , Antígenos CD28/inmunología , Complejo CD3/genética , Complejo CD3/inmunología , Antígenos CD4/genética , Antígenos CD4/inmunología , Linfocitos T CD4-Positivos/inmunología , Proliferación Celular , Citometría de Flujo , Perfilación de la Expresión Génica , Inmunidad Innata , Técnicas para Inmunoenzimas , Interleucina-10/genética , Interleucina-10/inmunología , Interleucina-12/genética , Interleucina-12/inmunología , Activación de Linfocitos/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Transgénicos , Monocitos/citología , Monocitos/inmunología , Análisis de Secuencia por Matrices de Oligonucleótidos , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Linfocitos T/inmunologíaRESUMEN
A number of important helminth parasites of humans have incorporated short-term residence in the lungs as an obligate phase of their life cycles. The significance of this transient pulmonary exposure to the infection and immunity is not clear. Employing a rodent model of infection with hookworm (Nippostrongylus brasiliensis), we characterized the long-term changes in the immunological status of the lungs induced by parasite infection. At 36 days after infection, alterations included a sustained increase in the transcription of both Th2 and Th1 cytokines as well as a significant increase in the number and frequency of alveolar macrophages displaying an alternatively activated phenotype. While N. brasiliensis did not induce alternate activation of lung macrophages in STAT6(-/-) animals, the parasite did induce a robust Th17 response in the pulmonary environment, suggesting that STAT6 signaling plays a role in modulating Th17 immunity and pathology in the lungs. In the context of the cellular and molecular changes induced by N. brasiliensis infection, there was a significant reduction in overall airway responsiveness and lung inflammation in response to allergen. In addition, the N. brasiliensis-altered pulmonary environment showed dramatic alterations in the nature and number of genes that were up- and downregulated in the lung in response to allergen challenge. The results demonstrate that even a transient exposure to a helminth parasite can effect significant and protracted changes in the immunological environment of the lung and that these complex molecular and cellular changes are likely to play a role in modulating a subsequent allergen-induced inflammatory response.
Asunto(s)
Pulmón/inmunología , Pulmón/parasitología , Nippostrongylus/inmunología , Alérgenos/inmunología , Animales , Citocinas/biosíntesis , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Activación de Macrófagos , Macrófagos Alveolares/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Datos de Secuencia Molecular , Análisis de Secuencia por Matrices de Oligonucleótidos , Factor de Transcripción STAT6/deficienciaRESUMEN
While it is well established that infection with the rodent hookworm Nippostrongylus brasiliensis induces a strongly polarized Th2 immune response, little is known about the innate host-parasite interactions that lead to the development of this robust Th2 immunity. We exploited the transient pulmonary phase of N. brasiliensis development to study the innate immune responses induced by this helminth parasite in wild-type (WT) and severe-combined immune deficient (SCID) BALB/c mice. Histological analysis demonstrated that the cellular infiltrates caused by N. brasiliensis transit through the lungs were quickly resolved in WT mice but not in SCID mice. Microarray-based gene expression analysis demonstrated that there was a rapid induction of genes encoding molecules that participate in innate immunity and in repair/remodeling during days 2 to 4 postinfection in the lungs of WT and SCID mice. Of particular note was the rapid upregulation in both WT and SCID mice of the genes encoding YM1, FIZZ1, and Arg1, indicating a role for alternatively activated macrophages (AAMs) in pulmonary innate immunity. Immunohistochemistry revealed that nearly all alveolar macrophages became YM1-producing AAMs as early as day 2 postinfection. While the innate responses induced during the lung phase of N. brasiliensis infection were similar in complexity and magnitude in WT and SCID mice, only mice with functional T cells were capable of maintaining elevated levels of gene expression beyond the innate window of reactivity. The induction of alternatively activated alveolar macrophages could be important for dampening the level of inflammation in the lungs and contribute to the long-term decrease in pulmonary inflammation that has been associated with helminth infections.
Asunto(s)
Pulmón/parasitología , Activación de Macrófagos/genética , Macrófagos Alveolares/inmunología , Nippostrongylus , Infecciones por Strongylida/inmunología , Animales , Arginasa/genética , Quimiocinas/genética , Citocinas/genética , Perfilación de la Expresión Génica , Inmunidad Innata/genética , Péptidos y Proteínas de Señalización Intercelular , Lectinas/genética , Pulmón/inmunología , Pulmón/patología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos , Ratones SCID , Factor de Crecimiento Nervioso/genética , Nippostrongylus/crecimiento & desarrollo , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas/genética , Infecciones por Strongylida/genética , Células Th2/inmunología , beta-N-Acetilhexosaminidasas/genéticaRESUMEN
A blood-stage vaccine based on Plasmodium falciparum merozoite surface protein 3 (MSP3) was tested for efficacy in a primate model. Aotus nancymai monkeys were vaccinated with yeast-expressed MSP3 before a lethal challenge with Plasmodium falciparum parasites. Five of 7 control monkeys had acute infections and required treatment to control parasitemia. Only 1 of 7 monkeys vaccinated with MSP3 required this treatment. The efficacy of the MSP3 vaccination appeared to be comparable to that of MSP1(42), a leading asexual vaccine candidate, in response to which 2 monkeys experienced acute infections. In the MSP3-vaccinated group, protection correlated with prechallenge titers of antibody to MSP3. In the MSP1 and control groups, protection correlated with antibody to MSP3 raised by challenge infection.